Minigene‐based splicing analysis and ACMG/AMP‐based tentative classification of 56 ATM variants

Abstract

The ataxia telangiectasia-mutated (ATM) protein is a major coordinator of the DNA damage response pathway. ATM loss-of-function variants are associated with 2-fold increased breast cancer risk. We aimed at identifying and classifying spliceogenic ATM variants detected in subjects of the large-scale sequencing project BRIDGES. A total of 381 variants at the intron-exon boundaries were identified, 128 of which were predicted to be spliceogenic. After further filtering, we ended up selecting 56 variants for splicing analysis. Four functional minigenes (mgATM) spanning exons 4-9, 11-17, 25-29, and 49-52 were constructed in the splicing plasmid pSAD. Selected variants were genetically engineered into the four constructs and assayed in MCF-7/HeLa cells. Forty-eight variants (85.7%) impaired splicing, 32 of which did not show any trace of the full-length (FL) transcript. A total of 43 transcripts were identified where the most prevalent event was exon/multi-exon skipping. Twenty-seven transcripts were predicted to truncate the ATM protein. A tentative ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme that integrates mgATM data allowed us to classify 29 ATM variants as pathogenic/likely pathogenic and seven variants as likely benign. Interestingly, the likely pathogenic variant c.1898+2T>G generated 13% of the minigene FL-transcript due to the use of a noncanonical GG-5'splice-site (0.014% of human donor sites). Circumstantial evidence in three ATM variants (leakiness uncovered by our mgATM analysis together with clinical data) provides some support for a dosage-sensitive expression model in which variants producing ≥30% of FL-transcripts would be predicted benign, while variants producing ≤13% of FL-transcripts might be pathogenic.

Comprehensive splicing analysis of the alternatively spliced CHEK2 exons 8 and 10 reveals three enhancer/silencer‐rich regions and 38 spliceogenic variants

“…Hybrid minigenes are simple versatile tools to initially assess the impact of any sequence variation on splicing, as we have previously shown in the main BC susceptibility genes [19][20][21][22][23][24]31,49]. In this regard, the maintenance of the genomic context, including intron length, is essential for exon recognition and, therefore, for minigene design [25,27,46].…”

“…We decided to assign a pathogenic (or benign) evidence strength only if ≥90% of the read-out signal supports pathogenic (or benign). We have previously applied the same conservative instance to complex read-outs observed in CHEK2 and other genes [22,23,31]. As a result, variants such as c.885A>G (p.Glu295=), expressing mostly canonical transcripts supporting benign (82% of the expression), end up as VUS because the remaining 18% of the signal supports pathogenic.…”

“…The binding of trans-acting factors to ESE sequences is essential for effective exon recognition, so that any variant overlapping these cis elements (including synonymous changes) may impair splicing and cause a genetic disorder [16,17]. In fact, RNA splicing disruption by germline variants is a prevalent mechanism of inactivation of the BC susceptibility genes [18][19][20][21][22][23][24].…”

Minigene Splicing Assays Identify 20 Spliceogenic Variants of the Breast/Ovarian Cancer Susceptibility Gene RAD51C

“…If neither pathogenic nor benign supporting transcripts contribute ≥90% to the overall expression, the splicing assay is considered to provide no evidence in favor of, or against, pathogenicity. Recently, we used a similar approach to deal with complex PALB2/ATM minigene readouts [ 20 , 30 ].…”

“…The rarity code PM2 was considered as per HBOPC_ATMv1 specifications ( , accessed on 2 May 2022) [ 30 ]: (i) allele frequency ≤ 0.01%; (ii) decreasing PM2 evidence strength to ‘supporting’. For allele counting, we interrogated gnomADv2.1 (global).…”